Knowledge of the structure and assembly of membrane-bound proteins lags behind the well-understood fundamental principles of soluble protein structure and folding. Genomic analysis suggests that approximately 20-30% of all proteins are membrane-bound, but less than 1% of the structures in the Protein Data Bank contain integral transmembrane domains. Few studies of membrane protein folding and assembly have been reported. The specific aims of this project are to test a hypothesis about the mechanism of transmembrane helix association, with the long-range goal of developing new methods for predicting three dimensional structures of transmembrane domains and for manipulating misfolded structures. A wide range of disease states, including cystic fibrosis, retinitis pigmentosa, and some demyelinating diseases involve misfolded membrane proteins. The hypothesis is that helical transmembrane proteins fold into their native conformation by first associating as specific helix pairs, which form templates for subsequent folding steps. Specific aim #1 : to examine the folding pathway of the membrane protein bacteriorhodopsin by equilibrium and kinetic methods, using the technique of fluorescence resonance energy transfer, with both natural and engineered fluorophore sites. The experiments will detect folding intermediates, if they exist, which will show whether nucleating helix-helix interactions occur. Specific aim #2: to determine whether associating transmembrane helices gain thermodynamic stability from the lipid bilayer, by examining the association of synthetic model peptides by fluorescence and cross-linking methods. Measurements will be made of the relative thermodynamic contributions to helix-helix association due to disordered lipid acyl chains and disordered protein side chains. Specific aim #3: to analyze new transmembrane protein structures for helix-helix association sites, using computational methods that identify potential smooth surfaces for association. These analyses may provide the basis for new structure-prediction algorithms for helical transmembrane proteins based on amino acid sequence information.